In the field of digital pathology, biological specimens such as tissue sections, blood, cell cultures and the like may be stained with one or more stains and analyzed by viewing or imaging the stained specimen. Observing the stained specimen, in combination with additional clinical information, enables a variety of processes, including diagnosis of disease, prognostic and/or predictive assessment of response to treatment, and assists in development of new drugs to fight disease. As used herein, a target or target object is a feature of the specimen that a stain identifies. A target or target object may be a protein, protein fragment, nucleic acid, or other object of interest recognized by an antibody, a molecular probe, or a non-specific stain. Those targets that are specifically recognized bay be referred to as biomarkers in this subject disclosure. Some stains do not specifically target a biomarker (e.g. the often used counterstain hematoxylin). Hematoxylin is a basic/positive compound that binds to and forms salts with acidic, or basophilic, compounds containing negative charges (such as DNA and RNA which are acidic/negative because the nucleic acid building blocks that come off the phosphate backbone are negatively charged) and stains them dark blue or violet. While hematoxylin has a fixed relationship to its target, most biomarkers can be identified with a user's choice of a stain. That is, a particular biomarker may be visualized using a variety of stains depending on the particular needs of the assay. Subsequent to staining, the assay may be imaged for further analysis of the contents of the tissue specimen. An image of an entire slide is typically referred to as a whole-slide image, or simply whole-slide.
Quantitative analysis of a whole-slide, such as counting target objects such as cells of a certain kind, or the quantitation of a staining response for all cells on a slide, is not feasible for human observers. Typically, a whole-slide contains several thousand to several hundred thousand cells, of which all or just a fraction may be relevant for an analysis question at hand. Methods from image analysis, computer vision, and pattern recognition can be used for an automated quantitative analysis.
One example of a whole slide image subject to image analysis is a cMET assay (also known as MET). MET is a receptor tyrosine kinase (RTK) known to be amplified, mutated or overexpressed in many solid malignancies, including non-small cell lung cancer (NSCLC). Abnormal MET activation in cancer correlates with poor prognosis, where aberrantly active MET triggers tumor growth, angiogenesis and metastasis. For example, the majority of squamous cell carcinoma (SQCC) expresses the protein product of Met mRNA at levels much lower than or similar to normal lung tissue or bronchial epithelium. Moreover, SQCC characteristically over-express a variant Met mRNA which corresponds to a 5′ partially deleted transcript produced by alternative splicing. In contrast, the expression of Met mRNA and its protein product in adenocarcinoma (ADC) and large cell undifferentiated carcinoma are heterogeneous: in approximately 35% and 20% of these subtypes of NSCLC, Met mRNA and its protein product is overexpressed. Among ADC, intermediate to high levels of Met immunoreactivity correlated with greater degree of tumor differentiation. Furthermore, an accentuation of Met immunoreactivity was often noted in cancer cells at the advancing edge of tumors. Thus, Met has been observed to play a role in lung cancer cell invasion and differentiation (Lung Cancer. 1998 April; 20(1):1-16: “Differential expression of Met/hepatocyte growth factor receptor in subtypes of non-small cell lung cancers”, Tsao M S1, Liu N, Chen J R, Pappas J, Ho J, To C, Viallet J, Park M, Zhu H).
The cMET assay stains the membranous and cytoplasmic region of the non-neoplastic and malignant cells. The categorization of MET expression in NSCLC is semi-quantitative and may comprise an evaluation of staining intensity and percentage positivity.
Manual assessment of these criteria is difficult or impossible, similar to detection and scoring of membranous and cytoplasmic regions in other IHC (“immunohistochemistry”)-stained tissue slides, for assays such as HER2 and EGFR, and for other cancerous tissue types, such as breast and gastric cancers.